Enzyme assay method using surface plasmon resonance spectroscopy

ABSTRACT

Method of performing enzyme assays using the technique of surface plasmon resonance spectrometry (SPRS). An SPRS surface carries an immobilized reagent which is capable of being released by an enzyme. A fluid sample containing the enzyme is brought into contact with the solid surface, and release of the reagent monitored by SPRS.

This invention concerns methods of performing enzyme assays using thetechnique of surface plasmon resonance spectrometry (SPSR).

The phenomenon of SPR is well known and will not be described in detail(see EPA 305109 for example). Briefly, the intensity of monochromaticplane-polarised light (conveniently obtained from a laser) reflectedfrom the interface between an optically transparent material, e.g.glass, and metal depends on the refractive index of material on thedownstream side of the metal. Accordingly, by measuring changes inintensity of reflected light an indication can be obtained of changes inrefractive index of material at a particular point on the downstreamsurface of the metal. The intensity of reflected light also varies withthe angle of incidence, and reflectively drops sharply to a minimum at aparticular angle which is characteristic of the equipment.

International Patent Application PCT/GB90/00432 filed Mar. 21, 1990makes use of this phenomenon in an enzyme assay. The solid surfacecarries thereon an immobilized enzyme which catalyses the reactionbetween two or more reactants resulting in the production of a reactionproduct, or a substrate for the enzyme. The method involves bringinginto contact with the solid surface a fluid medium containing theremaining reactants and if not already present the enzyme, underconditions to cause the reaction product to be deposited on the solidsurface. One molecule of enzyme can catalyze the deposition of manymolecules of reaction product on the solid surface. The change inrefractive index that results from deposition of the reaction product ismonitored by SPRS. That method provides a satisfactory assay for theenzyme, but one which requires the addition of one or more reagents tothe fluid medium.

This invention is a development of that method, and has the advantagethat no additional reagents are required, apart from a pretreated solidsurface and a liquid sample of the analyte.

a method of performing an assay for an enzyme according to the inventioninvolves the use of a solid surface carrying an immobilized reagentwhich is capable of being released by the enzyme. A fluid sample isbrought into contact with the immobilised reagent under conditions suchthat the enzyme if present in the sample catalyzes release of thereagent from the surface. The solid surface is provided by a metalliclayer applied to a block of material transparent to electromagneticradiation. Release of the reagent from the surface of the metallic layeris assayed by SPRS.

The immobilized reagent may be a substrate for the enzyme. Examples ofsuitable pairs of enzyme and substrate are DNAse/DNA; RNAse/RNA;amylase/starch; various glycosidases/their polysaccharide substrates;peptidases/polypeptides. In addition to these natural substrates,synthetic substrates can be made combining a molecule or particle (ofhigh or alternatively low refractive index with respect to the bulkphase) with the metal surface via an enzymatically sensitive linker thatis cleavable by the enzyme under study. Release of the molecule orparticle is then easily detected as a change in the SPRS signal.

As noted above, a major advantage of this technique is that it requiresno reagent other than the enzyme substrate pre-coated on the solidsurface of the SPR device. A further advantage is its directness. Thetechnique also meets a need--the classes and types of enzymes suited forthis approach are very difficult to assay directly and at highsensitivity by any other means.

It is not necessary that the enzyme be the primary analyte under study.There are very many assays in existence for a variety of analytes whichresult in the production of an enzyme in solution at a concentrationrelated to the concentration of primary analyte in a sample. Thisinvention provides a convenient and rapid technique for observing thepresence of concentration in a fluid of an enzyme from any source.

EXAMPLE

A glass slide coated with 50 nm thick silver-metal was placed on a fanbeam SPR instrument. The silver surface was coated by flowing about 0.5ml of 200 nM mouse anti-sheep immunoglobulin (MaS) made up in 10 mMphosphate buffer, pH 7.4, over the surface at a rate of 4 μl/sec. Thesurface was then washed by manual application of 3 ml of 10 mM phosphatebuffer, followed by the same volume of 10 mM borate buffer, pH 8.5.

Various concentrations of Pronase E (Protease XXV, from Streptomycesgriseus, 4.7 units/mg, Sigma Chemical Co.) as specified below wereprepared freshly in borate buffer, pH 8.5, immediately before use, bydilution from a stock of 1.4 mg/ml (equivalent to about 52 μM enzyme,made by dissolving 4.4 mg Pronase E in 3.1 ml borate buffer) that hadbeen prepared at the beginning of the day. The enzyme solution (about0.5 ml) volume was pumped over the surface at a flow-rate of 4 μl/sec.The base-line change after reaction was measured by performing a manualwash with borate buffer.

The buffer was then changed back to 10 mM phosphate, pH 7.4. Theresidual enzyme activity at the surface, and any exposed binding sitesat the surface were blocked by applying 3 ml of 0.5% bovine serumalbumin plus 2 μM human gamma-globulin (both from Sigma) made up in 10mM Phosphate, pH 7.4. The surface was washed with phosphate buffer, then0.5 ml of 1.5 μM ovine IgG (oIgG) was pumped onto the surface. Anyactive antibody remaining on the surface would be expected to bind thisprotein. The surface was finally washed twice with phosphate buffer toobtain the amount of protein specifically bound.

During these protein, buffer washes and blocking additions, the changein percent reflectivity was measured on the SPR instrument. This changeis directly related to the change in refractive index at up to 100 nmabove the silver surface (i.e. about ten layers of IgG molecules couldinfluence the SPR signal). The enzyme activity was calculated as theinitial rate of change in percent reflectivity per second. The amount ofmouse anti-sheep immuno-globulin bound at the surface was monitored bymeasuring the difference in percent reflectivity (%R) after and beforeprotein addition (Δ % R_(MaS)). Similarly, the amount of ovine IgG boundwas the difference between percent reflectivity after and before proteinaddition (Δ % R_(oIgG)). The results of the ovine IgG bound to the mouseanti-sheep immunoglobulin, to account for the difference between silverslides, was normalized by taking the ratio of Δ % R_(oIgG) /Δ % R_(MaS).The fraction of mouse anti-sheep immunoglobulin removed was calculatedas (1-(change in % reflectivity sheep-IgG)/(change in % reflectivityMaS).

    ______________________________________                                        Results                                                                       ______________________________________                                        Enzyme Concentration (nM)                                                                     80       200        220                                       Rate of Hydrolysis                                                                            0.0038   0.0073     0.0098                                    (-Δ % R/sec)                                                            Fraction of MaS Removed                                                                       0.31     0.66       0.77                                      (1 - Δ % R.sub.oIgG /Δ % R.sub.MaS)                               ______________________________________                                    

1. Protease hydrolysis of protein bound at the SPR surface.

A graph of these data gives a straight line passing through the origin,as would be expected for an enzyme rate is dependent on concentration.

2. Protease hydrolysis rate against the fraction of protein removed.

The enzymic reaction rate correlates with the fraction of antibodyremoved from the surface, as determined by back-titration of activeantibody by measuring the binding of added ovine IgG. A straight linecorrelation that passes through the origin is expected.

We claim:
 1. A method of performing an assay for an enzymecomprising:providing a solid surface carrying an immobilized reagentwhich is capable of being released by the enzyme; contacting theimmobilized reagent with a fluid sample under conditions such that theenzyme, if present in the fluid sample, catalyzes release of theimmobilized reagent from the solid surface; and detecting release of theimmobilized reagent from the solid surface by surface plasmon resonancespectroscopy thereby determining the presence of the enzyme, wherein thesolid surface is a metallic layer applied to a block of materialtransparent to electromagnetic radiation.
 2. The method as claimed inclaim 1, wherein the reagent is a substrate for the enzyme.
 3. Themethod as claimed in claim 1, wherein the reagent comprises a moleculeor particle, of high or low refractive index with respect to the fluidsample, joined to the surface by means of a bond which is cleavable bythe enzyme.
 4. The method as claimed in claim 1, wherein the reagentcomprises a substrate for the enzyme, such that the enzyme and thesubstrate constitute an enzyme/substrate pair is selected from the groupconsisting of DNAse/DNA; RNAse/RNA; amylase/starch;glycosidases/polysaccharides; and peptidases/polypeptides.
 5. The methodas claimed in claim 4, wherein the enzyme is a peptidase and the reagentcomprises a polypeptide.